For an organism to survive, its proteins must adopt a diversity of conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. This situation becomes critical when considering proteins with energetic folding landscapes that permit many conformational states. In these cases, the environment can clearly influence the conformation by favoring one pathway over another. Because the aggregating proteins that are responsible for neurodegenerative diseases like Alzheimer?s and Parkinson?s diseases often have identical sequences in healthy and diseased individuals, differences in cellular environment are responsible for the conformational switch. Yet, despite the importance of the environment for protein folding, structural investigations of biomolecules are typically confined to in vitro systems, which cannot capture important structural features imposed by biological environments. Solid-state NMR spectroscopy is currently undergoing a ?sensitivity renaissance? with the development of dynamic nuclear polarization (DNP). Experiments that would require decades of experimental time with traditional ssNMR methods can be collected in a day with DNP NMR. The proposed work seeks to advance our knowledge about protein structure in natural and disease-relevant biological contexts though continued development of the methodology for in-cell structural biology using sensitivity-enhanced NMR spectroscopy and the application of these approaches to cellular and whole organism models of neurodegenerative diseases, tightly coupling protein structures with genotype, phenotype and environment.
The misfolded proteins associated with neurodegenerative disease can adopt a variety of different conformations, some of which are toxic. Because these proteins have identical amino acid sequences, the cellular environment clearly influences the final state, yet most structural studies do not include the cellular context and, perhaps because we are not studying the correct conformation, not a single therapeutic strategy for these diseases addresses the underlying protein misfolding pathology. Using new sensitivity-enhancement technology for solid state NMR spectroscopy, we will study protein structure in native environments - inside living cells - to reveal how both healthy and disease-relevant cellular environments influence protein structure.
